Study On Gut Microbiota And Interleukin-17 Characteristics Of Rhesus Macaques

BackgroundMucosa is known as the main portal for pathogens including human immunodeficiency virus.HIV-1 infection caused severe results,including massive CD4+T cell depletion in mucosa,destruction of intestinal epithelial cells,microbial translocation and sustained immune activation.Studies have demonstrated that microbial translocation can stimulate the immune system to cause systemic immune activation,further aggravating the inflammatory response and disease progression in HIV-1 patients.In addition,Th17 cells were selectively cleared as a result of the significant reduction of mucosal CD4+T cell and IL-17A as the characteristic cytokine of Th17 cells was abnormally expressed in HIV/SIV-infected intestinal mucosa.The gut microbiota had been closely related to mucosal Th17 cells.However,there are few reports about whether the change of gut microbiota in AIDS patients is related to the change of IL-17.Furthermore,IL-17A and IL-17F play important roles in autoimmune diseases,infections and immunity.These findings are mostly obtained in mouse models.Rhesus macaque,as a crucial animal model for human diseases(especially HIV),can play an important role in the relationship between AIDS-related gut microbiota changes and IL-17 changes as well as other IL-17-related diseases.The biological activity of IL-17A and IL-17F and gut microbita of rhesus macaques are still poorly understood.This study systematically analyzed the diversity,relative abundance of taxa composition and functional genes of the rectal microbiota of rhesus monkeys;compared the differences of rectal microbiota between rhesus macaques and cynomolgus macaques and african green monkeys in diversity,relative abundance of taxa composition and functional genes;analyzed the effects of external factors such as rectal immunization,oral vancomycin and SHIV/SIV infection on gut microbiota;identified the molecular and biological characteristics of rhesus macaques IL-17A and IL-17F and preliminary analysed the correlation between gut microbiota and the expression of IL-17A and IL-17F.Methods1.Deep sequencing of 16S rRNA V4 region method was established to sequence the rectal samples of 20 rhesus macaques,21 cynomolgus macaques and 20 african green monkeys,as well as 197 rectal washing samples from immunization experiments,30 rectal samples from oral vancomycin experiments and 10 duodenum mocosa samples of SHIV/SIV infection of rhesus macaques.QIIME2,PICRUStl and STAMP software were used for bioinformatics analysis?2.Real-time quantitative RT-PCR was established to detect the IL-17A and IL-17F mRNA levels in PBMC,rectal mocosa of vancomycin experiments and duodenum mocosa of SHIV/SIV infection experiments of rhesus macaques.Statistical methods were used to analyze the correlation between related microbiota and IL-17A/IL-17F mRNA levels.3.Real-time quantitative PCR was established to detect the SFB(segmented filamentous bacteria)and BCCT(Butyryl-CoA:acetate CoA-transferase)DNA levels in rectal fecal between rhesus macaques and cynomolgus macaques and african green monkeys.Statistical methods were used to analyze the correlation between microbiota of three species and SFB and BCCT DNA levels.4.The method to separate the gut microbiota in the rectal fecals of rhesus macaques,cynomolgus macaques and african green monkeys was established and tested the effects of the gut microbiota of three monkeys on the expression of tight junction-related genes,inflammatory factors and proinflammatory factors5.RACE amplification methods were used to clone the nucleotide sequences of IL-17A and IL-17F cDNA.The full-length cDNA of rhesus macaque IL-17A and IL-17F was obtained by sequencing.Real-time quantitative RT-PCR was used to analyze the expression of IL-17A and IL-17F in different tissues of normal rhesus macaques.Purified recombinant rhesus macaque IL-17A and IL-17F proteins with functional activity were obtained through prokaryotic expression,purification,and endotoxin removal.6.Statistical analysisFor quantitative comparison of the diversities and compositions of the rectal microbiota between different groups,Mann-Whitney test,ANOSIM or Welch's t-test were used.For fluorescence quantitative data analysis,Mann-Whitney test or unpaired two-tailed t test were used.For correlation analysis,Spearman test was used.P-values less than 0.05 were considered statistically significant.Results1.Bacteroidetes,Firmicutes and Proteobacteria were dominant phyla in rhesus macaques.Prevotellaceae,Ruminococcaceae,[Paraprevotellaceae],Lachnospiraceae and Veillonellaceae were dominant family in rhesus macaques.Prevotella,Oscillospira,[Prevotella],Streptococcus and Faecalibacterium were dominant genus in rhesus macaques.Prevotella copri,Streptococcus luteciae,Faecalibacterium prausnitzii and Prevotella stercorea were dominant species in rhesus macaques.Further analysis found that gender could influence the composition and functional genes of microbiota of rhesus macaques and group size could influence the obseved group difference.In addition,the predominant phylum of rhesus macaques is consistent with human and there are slight differences in the predominant family and genus.Bacteroidaceae and Bacteroides are dominant family and genus in human rectal microbiota.2.The dominant taxa of rhesus macaques,cynomolgus macaques and african green monkeys at the phylum,family,genus and species levels are consistent.Further analysis found that gender can influence the composition and functional genes of microbiota of cynomolgus macaques and african green monkeys.3.The alpha diversity of rectal microbita of rhesus macaques was the highest.Although the dominant taxa of rectal microbiota of rhesus macaques,cynomolgus macaques and african green monkeys were the same,the relative abundance of dominant taxa were significantly different.The relative abundance of Firmicutes of rhesus macaques was the highest compared to cynomolgus macaques and african green monkeys,whereas the relative abundance of Bacteroidetes in african green monkey was the highest.Metagenomics prediction found that there were many differences of rectal microbiota of the three species,including differences in lipopolysaccharide(LPS)biosynthesis proteins,LPS biosynthesis,Transporters and ABC transporters.4.The expression of SFB DNA levels in rectal fecal of rhesus macaques was significantly lower than that of cynomolgus macaques and slightly higher than that of african green monkeys;the expression of BCCT DNA levels of rhesus macaques was lower than that of african green monkey and cynomolgus macaques without statistically significant.The rectal microbiota of rhesus macaques,as well as cynomolgus monkeys and african green monkeys,had little effect on the expression of tight junction-related genes,however,the rectal microbiota of african green monkey induced the lowest level of inflammatory factors expression.5.Rectal immunization could influence the diversity and reduce the relative abundance of Firmicutes and increase the abundance of Bacteroidetes of rhesus macaques.At the same time,the expression levels of functional genes of pathways related to nucleotide metabolism and protein synthesis were significantly increased.Oral vancomycin could influence the diversity and reduce the relative abundance of Bacteroidetes and increase the relative abundance of Firmicutes as well as significantly increasing functional genes related to Transporters and ABC Transporters of the rectal microbiota of rhesus macaques.SHIV/SIV infection had little effect on the diversity,but significantly reduced the relative abundance of Anaerovibrio,Parabacteroides,Gemmiger,Dialister,Methyloversatilis and Prevotella copri of the duodenal mucosa microbiota of rhesus macaques.Furthermore,SHIV/SIV infection significantly increased the functional genes in pathways related to lipid biosynthesis proteins,amino acid metabolism and energy metabolism.6.The full length of IL-17A cDNA is 1283bp(43 bp-5'UTR,468 bp-ORF and 772 bp-3'UTR).The full length of IL-17F cDNA is 820bp(72bp-5'UTR,492bp-ORF and 256bp-3'UTR).The genomic mapping revealed that the IL-17A and IL-17F gene contains three exons and two introns,locates on chromosome 4 with the two cytokines in adjacent positions.The nucleotide sequence and amino acid sequence of rhesus IL-17A gene shows 95.79%and 96.8%similarity with that of human IL-17A respectively.The nucleotide sequence and amino acid sequence of rhesus IL-17F gene shows 96.23%and 93.9%similarity with that of human IL-17F respectively.In addition,the conserved sites closely related to the structure and function of rhesus IL-17A and IL-17F such as cysteine and N-glycosylation sites are all consistent with that of human IL-17A and IL-17F.7.Both IL-17A and IL-17F are highly expressed in lymph node,spleen,tonsil,thymus and the intestinal tract as compared to the other tissues examined.Rhesus macaque IL-17F mRNA exhibited a higher level in lung,heart,liver,kidney and pancreas compared to IL-17A mRNA.In addition,high levels of IL-17A and IL-17F mRNA can also be detected in the bronchus and vagina tissues.8.Rhesus macaque IL-17A and IL-17F showed similar bioactivities compared to that of human IL-17A and IL-17F,such as activating the phosphorylation of NF-?B-p65,inducing the production of anti-bacterial proteins(?-defensin 2,S100A8,S100A9,RegIII? and Muc1),upregulating the expression of tight junction associated genes(CLDN1,CLDN4,OCLN and ZO-1)of Caco-2 cells and inducing the expression of IL-6 and TNF-? by THP-1 cells.9.IL-17F mRNA levels in PBMC was significantly positively correlated with the relative abundance of Fibrobacteres of rectal microbiota of rhesus macaques.IL-17F mRNA levels in duodenal mucosa of rhesus macaques was significantly negatively correlated with the relative abundance of Bacteroidetes and positively correlated with the relative abundance of Cyanobacteria.The IL-17A mRNA in rectal mocosa was positively correlated with the relative abundance of Bacteroidetes and negatively correlated with the relative abundance of Firmicutes without statistical differences.ConclusionThe predominant phylum Bacteroidetes and Firmicutes of rhesus macaques is consistent with human and there are slightly differences in the predominant family and genus.The rectal microbiota of rhesus macaques had significant differences in diversity,taxa composition,predicted functional genes and in vitro functions compared to cynomolgus macaques and african green monkeys.Rectal immunization,oral vancomycin and SIV/SIV infection could influence the gut microbiota of rhesus macaques.In addition,the sequence characteristics and tissue distribution of rhesus macaque were the same with human IL-17A and IL-17F.Recombinant rhesus IL-17A and IL-17F could induce the phosphorylation of NF-?B-p65,up-regulate the expression of antibacterial peptides and tight junction protein-related genes and promote the release of proinflammatory cytokines.Furthermore,the IL-17A/IL-17F mRNA levels in PBMC and intestinal mocosa were correlated with gut microbiota of rhesus macaques.These results showed that the gut microbiota,IL-17A and IL-17F of rhesus macaques are similar to that of human.In conclusion,our study have laid a foundation for further study on the mechanism of the interaction between gut microbiota and IL-17 in HIV/AIDS,testing the gut microbiota and the effect of targeting IL-17 in HIV/AIDS therapy.